It is known that dissolved carbon dioxide alone or in combination with some other factors like osmolality, glucose concentration, or pH can effect cell growth and performance (Zanghi et al., 1999; Takuma et al., 2007). All publications describing methods utilising dissolved carbon dioxide to regulate cell growth report on a CO2 partial pressure (pCO2) that is kept constant throughout the whole bioprocess.
Zanghi et al. (1999) have further focused on the effect pCO2 has on glycosylation of recombinant proteins, and concluded that under elevated pCO2 the intracellular pH can be affected which leads to decreased activity of pH-sensitive sialyltransferases and consequently to decreased polysialylation of recombinant proteins.
Takuma et al. (2007) have found that the sugar mapping profile was significantly affected by glucose concentrations, where at higher glucose conditions the relative bG0 peak area was higher, while on the other hand they did not find a consistent dependence on pCO2.
Only two references were found to mention a strategy for influencing the degree of glycosylation and the glycosylation profile in the product by dissolved CO2 in cell culture bioprocesses. Whereas one demonstrates a negative correlation between pCO2 and product polysialylation (Zanghi et al., 1999), the other (Takuma et al. 2007) concludes that no consistent dependence of glycan structures bG0 and bG1 on pCO2 can be found.
Protein glycosylation typically leads to a diversity of proteins distinguished by their glycosylation profiles (patterns), which diversity is due to multiple factors. The question of native versus foreign glycosylation in humans is a contentious subject: It is not totally clear which state is the native glycosylation state, since it varies frequently between different and even within one species. This is especially true in IgG, the backbone molecule of most therapeutic monoclonal antibodies, but likewise applies to many other (types of) polypeptides and proteins.
Even with knowledge of the glycosylation profile of a particular human glycoprotein, matching those attributes of the recombinant protein produced in a bioprocess is not trivial. The choice of the host cell as the expression system has a primary role towards the resulting glycosylation profile of the respective polypeptide.
The three most common cell culture production modes are batch, fed-batch, and perfusion. The production method can have a pronounced effect on the resulting glycosylation profile. The glycosylation profile of the reporter protein-secreted alkaline phosphatase (SEAP) produced by a CHO DG44 cell line was compared between different process parameters: unamplified versus MTX-amplified cell lines, batch mode versus repeated fed-batch mode versus semi-continuous perfusion mode (Lipscomb et al., 2005). The glycosylation profile of the MTX-amplified cell line exhibited less mannosylated polypeptides, as well as less overall sialylation, although these differences were less than 10% of the total profile (that is, more than 90% of the sugar moieties attached to the polypeptides were the same and at the same position, regardless which process parameters had been selected). Overall sialylation was increased in the perfusion mode cultures compared to fed-batch mode, the slower growing cells in perfusion mode facilitated a more fully glycosylated polypeptide compared to the fed-batch mode where cells grew faster.
Researchers investigated the effect of various process parameters on the resulting EPO-Fc glycoprotein (EPO=erythropoietin) expressed by CHO cells in a bioreactor (Trummer et al., 2006). It was found that the sialic acid ratio (i.e., molar ratio NANA/glycoprotein; NANA=N-acetylneuraminic acid) in the glycoprotein had a maximum of about 13% when cultivated at around pH 7.0, which ratio decreased if the pH deviated therefrom. The optimal level of dissolved oxygen (DO) for the maximum ratio was accomplished by 50% of air saturation (the relationship pH/DO is that the DO level may indirectly interfere with pH, since pH is controlled by CO2/base, and CO2 addition can cause stripping of O2 from the vessel; e.g., high DO levels and low pH set-points can cause big oscillations in pH and pO2).
Similarly, the sialic acid ratio decreased from 13 to only 8% when the culture temperature was brought down from 37° C. to 30° C. This process mapping is a fine example of range-finding efforts that are pivotal towards process understanding and effective process control and its impact on protein glycosylation. DO levels were monitored continuously throughout the process of culturing mammalian cells and have been shown to affect the glycosylation profiles. Hypoxia in bioreactor cultures has shown ambiguous effects on protein glycosylation. For example, only minor changes were observed for tPA glycosylation, but significant sialylation changes were observed for FSH, although both proteins were produced by CHO cells. An IgG1 glycoprotein, expressed by a murine hybridoma at DO levels of 100, 50, and 10%, had a degree of galactosylation decreasing with the DO level.
Ammonium ions are a cellular waste product, generally toxic to cells, and accumulate in cell culture media principally as a result of glutamine and asparagine metabolism Ammonium chloride causes an increase in intracellular pH. Since terminal glycosylation occurs in the acidic distal regions of the Golgi complex, an increased level of ammonium chloride in the medium may, via an intracellular pH increase, correlate with a decrease in terminal sialylation.
The pH of the medium in the bioreactor has also been found to affect glycosylation profiles. The pH of a hybridoma culture has been shown to affect the resulting galactosylation and sialylation of the antibody. The highest levels of a-galacto- and mono-galacto-complex-type N-glycans were measured at pH ranging from 7.2 to 6.9, and the highest di-galacto-complex-type N-glycans were measured at pH 7.4, both in HEPES-buffered cultures. The latter condition also facilitated the highest NANA/NGNA (NGNA=N-glycolylneuraminic acid) ratio, compared to any of the pH experiments.
The proportion of acidic isoforms of EPO increased with decreasing pH of the cell culture, with optimal ranges of 6.8-7.2 favouring sialylation. Interestingly, even though higher pH and higher buffered conditions facilitated higher NANA contents, the opposite was found with polysialic acid attached to neural cell adhesion molecules (NCAM) expressed on recombinant CHO cell surfaces.
Reducing the temperature can increase overall polypeptide production by prolonging cell viability which should basically improve glycosylation (generally, improved glycosylation means a glycosylation profile coming closest to the profile of the originator protein/antibody; here, improved glycosylation means a higher degree of glycosylation). Cell viability is critical because extracellular glycosidases can accumulate in the medium and step-wise remove monosaccharides from the glycoprotein. Temperature shifts in a bioreactor can increase the product titre while maintaining glycoform quality (i.e., distribution of the glycoforms, in %). In contrast, EPO-Fc had a decrease in sialylation by 20% and 40%, when reducing the temperature to 33° C. and 30° C., respectively (Trummer et al., 2006). In this specific case, a reduced temperature of 30° C. showed a correlation between increased specific productivity and decreased levels of sialylation. It is still unknown, however, whether higher productivity correlates with the expression rate, thereby reducing the intracellular processing time for glycosylation and causing an increase in less sialylated protein populations. High cell viability (less sialidase activity) in conjunction with high cell productivity (shorter residence time) may diminish the overall effect on sialylation, i.e., cause sialylation to decrease.
Finally, shear stress in a bioreactor culture has been reported to be an important parameter to determine the resulting glycosylation profile. By manipulating agitation speeds and the resulting shear stress, it was found that maximum levels of damaging shear were required to minimise the extent of carbohydrate attachment to Asn184 in tPA, which was attributed to a decreased residence time of tPA in the ER. This can be an important consideration during scale-up or transfer of an existing process to a different facility. Monitoring the effects of shear differences on protein glycosylation during the transition between production modes (e.g., from perfusion to fedbatch) is also important for ensuring product comparability.
The above process parameters and further factors potentially having an impact on the glycosylation profile of a given polypeptide are a challenge for biopharmaceutical companies, where the change of the cell line, of process parameters, and/or of manufacturing site(s) may require extensive comparative studies to prove that the molecule remains the same. This was highlighted in the case of Genzyme's recombinant enzyme Myozyme. Genzyme was unable to demonstrate by FDA standards that Myozyme had the same carbohydrate structure when transferring the manufacturing process from the 160- to the 2000-liter bioreactor scale, underlining how critical it is to understand what affects the glycosylation profile.
Thus, in order to achieve comparability (within the meaning of similarity or even identity) between polypeptide products produced by different companies and/or via different methods (e.g., between a product of an originator and the corresponding product, i.e., the biosimilar, of another company), a method for controlling the glycosylation profile, posttranslational modifications manifested in CEX (cationic exchange chromatography) profiles, and variability between bioprocesses (variability also including growth profiles, metabolism, i.e., substrate consumption and metabolite production, product formation) would be extremely straightforward and desired.
In particular, the principal object underlying the present invention is to provide a method that allows to control the glycosylation profile and acidic variants in products produced in bioreactors (e.g., recombinant antibodies, cytokines, enzymes, hormones, growth factors) by only one parameter that is optionally easily set and regulated. Accordingly, such method would contribute to and enrich the art quite significantly.